Abstract
Microsomes were prepared from 20 day old Bomi barley endosperm by sucrose density gradient centrifugation. Electron microscopy revealed the presence of ribosome-studded vesicles in the material banding at the 1.75–2.26m sucrose interface. The isolated microsomes were active in the wheat-germ cell-free protein synthesizing system. Hordeins were present among the in vitro synthesized products and were identified by their solubility in 55% isopropanol and by their co-migration with native hordeins on SDS-polyacrylamide gels. The microsome-and polysome-directed, in vitro synthesized hordeins were analysed before and after chymotrypsin treatment by SDS-polyacrylamide gel electrophoresis. The hordeins were degraded when polysomes were used as a template. However, hordeins synthesized on microsomes showed significant protection from proteolysis. This protection could be abolished by treatment with membrane-solubilizing detergents. The reported experiments show that hordeins synthesized on microsomes were discharged vectorially into the lumen of the microsomes.
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Blobel, G. &B. Dobberstein: Transfer of proteins across membranes. I. Presence of proteolytically processed and nonprocessed nascent immunoglobulin light chains on membrane-bound ribosomes of murine myeloma. J. Cell Biol. 67, 835–851 (1975)
Blobel, G. &B. Dobberstein: Transfer of proteins across membranes. II. Reconstitution of functional rough microsomes from heterologous components. J. Cell Biol. 67, 852–862 (1975)
Bonner, W. M., &R. A. Laskey: A film detection method for tritium-labelled proteins and nucleic acids in polyacrylamide gels. Eur. J. Biochem. 46, 83–88 (1974)
Brandt, A. &J. Ingversen:In vitro synthesis of barley endosperm proteins on wild type and mutant templates. Carlsberg Res. Commun. 41, 312–320 (1976)
Burke, G. T. &C. M. Redman: The distribution of radioactive peptides synthesized by polysomes and ribosomal subunits combinedin vitro with microsomal membranes. Biochem. Biophys. Acta 299, 312–324 (1973)
Burr, B. &F. A. Burr: Zein synthesis in maize endosperm by polyribosomes attached to protein bodies. Proc. Nat. Acad. Sci. USA 73, 515–519 (1976)
Christianson, D. D., U. Khoo, H. C. Nielsen &J. S. Wall: Influence ofopaque-2 andfloury-2 genes on formation of proteins in particulates of corn endosperm. Plant Physiol. 53, 851–855 (1974)
Chua, N.-H. &P. Bennoun: Thylakoid membrane polypeptides ofChlamydomonas reinhardtii wild type and mutant strains deficient in photosystem II reaction center. Proc. Nat. Acad. Sci. USA 72, 2175–2179 (1975)
Dobberstein, B., G. Blobel &N.-H. Chua:In vitro synthesis and processing of a putative precursor for the small subunit of ribulose-1,5-bisphosphate carboxylase ofChlamydomonas reinhardtii. Proc. Nat. Acad. Sci. USA 74, 1082–1085 (1977)
Highfield, P. E. &R. J. Ellis: Synthesis and transport of the small subunit of chloroplast ribulose bisphosphate carboxylase. Nature 271, 420–424 (1978)
Ingversen, J.: Structure and composition of protein bodies from wild-type and high-lysine barley endosperm. Hereditas 81, 69–76 (1975)
Kreibich, G. &D. D. Sabatini: Microsomal membranes and the translational apparatus of eukaryotic cells. Fed. Proc. Fed. Amer. Soc. Exp. Biol. 32, 2133–2138 (1973)
Larkins, B. A., C. E. Bracker &C. Y. Tsai: Storage protein synthesis in maize. Isolation of zein-synthesizing polyribosomes. Plant Physiol. 57, 740–745 (1976)
Luthe, D. S. &D. M. Peterson: Cell-free synthesis of globulin by developing oat (Avena sativa). Plant Physiol. 59, 836–841 (1977)
Marcu, K. &B. Dudock: Characterization of a highly efficient protein synthesizing system from commercial wheat germ. Nucleic Acids Res. 1, 1385–1397 (1974)
Munck, L. &D. von Wettstein: Effects of genes that change the amino acid composition of barley endosperm. In: Genetic improvement of seed proteins. Proc. of a workshop 18–20 March 1974. National Academy of Sciences, Washington D. C. pp. 71–82 (1976)
Roberts, B. E. &B. M. Paterson: Efficient translation of tobacco virus RNA in a cell-free system from commercial wheat germ. Proc. Nat. Amer. Soc. USA 70, 2330–2334 (1973)
Shore, G. C. &R. Harris: Fate of polypeptides synthesized on rough microsomal vesicles in a messenger-dependent rabbit reticulocyte system J. Cell Biol. 74, 315–321 (1977)
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Cameron-Mills, V., Ingversen, J. & Brandt, A. Transfer of in vitro synthesized barley endosperm proteins into the lumen of the endoplasmic reticulum. Carlsberg Res. Commun. 43, 91 (1978). https://doi.org/10.1007/BF02906506
DOI: https://doi.org/10.1007/BF02906506